A promoter is a DNA sequence that promotes gene transcription to produce a messenger RNA and is recognized by RNA polymerase, which the promoter directs to the correct transcriptional start site. The E. coli promoter region, which is about 50 bases long, includes a common sequence (Pribnow box) of about 6 bases located upstream (in the 5' direction) from the site at which RNA synthesis begins. Five to eight bases separate the Pribnow box from the initiation site, so that it centers around the -10 position. Not all E. coli promoters have exactly the same sequence of bases in this region, but a sequence similar to TATAAT is typically found. Another consensus sequence, TTGACA, centers around -35; this region is also critical for the accurate and rapid initiation of transcription for most bacterial genes (see Darnell et al., 1986, Molecular Cell Biology, Scientific American Books Inc.). It has been noted that the -10 and -35 regions, as well as the spacing between them, are "major determinants of promoter strength." Because of the conserved regions, the RNA polymerase appears to make direct contact with the two regions. Consequently, "most mutations that affect promoter strength map to the strongly conserved bases in the -10 or -35 region or change the spacing between them" (Jansen, et al., 1985, Microbiology, Meetings for the American Society for Microbiology, American Society for Microbiology, Washington, D.C., pages 392-396 [1985]).
Mutations in the promoter may effect initiation. It has been shown that changes, deletions, or insertions in the promoter region may effect the way that RNA polymerase binds to the promoter. The ultimate result may be that more or less mRNA is produced. For example, Mandecki et al., 1982, Nucleic Acid Research 10:903-912, show that a two base pair insertion between the -10 and -35 regions lowered the promoter activity to 15% of the wild type; Post et al., 1978, Cell 15:231-236, show that a point mutation six bases upstream from the in vitro transcription site, which changed the Pribnow box, was important in the expression of an operon; and Youderian et al., 1982, Cell 30:843-853, show that specific decreases in the homology of a specific promoter with it consensus promoter sequence resulted in either a severe or a mild reduction in promoter activity (see Watson et al. Molecular Biology of Gene 4th edition Benjamin/Cummings Publishing Company, 1987). (Also see Botstein, 1972, Virology 49:268-282, and Siebenilist et al., 1980, Cell 20:269-281.)
Since promoters are known to effect ultimate gene expression, it would advantageous to increase the strength of promoter by manipulating the promoter sequence. That is the focus of the present invention, which is detailed and described more fully below.